VISUALIZATION OF PSEUDOCAVERNS IN STIRRED VESSELS

A new technique for reconstructing the geometry of well-mixed regions (pseudocaverns for Newtonian fluids) in unbaffled vessels agitated with radial discharge impellers eccentrically placed has been developed for obtaining their three dimensional geometry. The mixer frame rotates at low speed allowing the video system to capture a sequence of pictures of the flow patterns by using the laser induced fluorescence technique. The recorded video sequence was used to isolate the contours of the pseudocaverns for reconstructing the three dimensional structure of the well-mixed regions within the tank

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

Prova de tipográfica (In Press).Aims: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. Methods and Results: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. ANOVA indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. Conclusions: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. Significance and Impact of the Study: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error

Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1×106?1×109 cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our ?Region of Interest? method, with the software's ?Standard Area Method? of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 ?g/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Strategies for locating the female gamete: the importance of measuring sperm trajectories in three spatial dimensions

The spermatozoon must find its female gamete partner and deliver its genetic material to generate a new individual. This requires that the spermatozoon be motile and endowed with sophisticated swimming strategies to locate the oocyte. A common strategy is chemotaxis, in which spermatozoa detect and follow a gradient of chemical signals released by the egg and its associated structures. Decoding the female gamete’s positional information is a process that spermatozoa undergo in a three-dimensional (3D) space; however, due to their speed and small size, this process has been studied almost exclusively in spermatozoa restricted to swimming in two dimensions (2D). This review examines the relationship between the mechanics of sperm propulsion and the physiological function of these cells in 3D. It also considers whether it is possible to derive all the 3D sperm swimming characteristics by extrapolating from 2D measurements. It is concluded that full insight into flagellar beat dynamics, swimming paths and chemotaxis under physiological conditions will eventually require quantitative imaging of flagellar form, ion flux changes, cell trajectories and modelling of free-swimming spermatozoa in 3D

Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 198 (2012): 1075-1091, doi:10.1083/jcb.201204024.Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca2+ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca2+ response was produced. Additional molecules delivered during a Ca2+ response reset the cell by causing a pronounced Ca2+ drop that terminated the response; this reset was followed by a new Ca2+ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.This work was supported by the German Research Foundation and the Fonds der Chemischen Industrie.2013-03-1

Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expected

Summary: • Glomus intraradices is a widespread arbuscular mycorrhizal fungus (AMF), which has been found in an extremely broad range of habitats, indicating a high tolerance for environmental factors and a generalist life history strategy. Despite this ecological versatility, not much is known about the genetic diversity of this fungal species across different habitats or over large geographic scales. • A nested polymerase chain reaction (PCR) approach for the mitochondrial rRNA large subunit gene (mtLSU), distinguished different haplotypes among cultivated isolates of G. intraradices and within mycorrhizal root samples from the field. • From analysis of 16 isolates of this species originating from five continents, 12 mitochondrial haplotypes were distinguished. Five additional mtLSU haplotypes were detected in field‐collected mycorrhizal roots. Some introns in the mtLSU region appear to be stable over years of cultivation and are ancestral to the G. intraradices clade. • Genetic diversity within G. intraradices is substantially higher than previously thought, although some mtLSU haplotypes are widespread. A restriction fragment length polymorphism approach also was developed to distinguish mtLSU haplotypes without sequencing. Using this molecular tool, intraspecific genetic variation of an AMF species can be studied directly in field plants

Digital image analysis for microscope characterisation of the critical parameters involved in the fermentative production of secondary metabolites

Secondary metabolites, particularly antibiotics, are among the most relevant pharmaceutical compounds worldwide and they are generally produced by submerged cultivation of filamentous fungi. From the hydrodynamical point of view, these processes deal with the dispersion of up to four different phases: water-oil-air-fungi. It is well recognized that hydrodynamics has a significant influence on the global performance of the fermentation process; however, very little is understood about the relationships between hydrodynamics-dispersion-physiology-productivity. This poor understanding is partially due to the lack of precise methodologies to assess fungal viability (determining both the yield and productivity of the metabolite) and the size of the air bubbles and oil drops (related to the nutrient transport efficiency like oxygen and fatty acids). In this work, the published developments of our group, regarding image analysis-based methodologies for the study of Trichoderma harzianum cultivation for antimycotics production (v.g. 6-pentyl-alpha-pyrone) have been summarized and revised. The application of such methodologies resulted in the improvement of basic knowledge of the phenomena occurring within the fermenter as well as in the development of strategies for increasing the productivity of these processes. Key words: micelial cultures; hydrodynamics; multiphasic dispersión; microbial physiology; productivity; image analysis.Los metabolitos secundarios, y particularmente los antibióticos, se encuentran entre el grupo de fármacos de mayor relevancia en el mercado mundial, y son producidos, en su mayoría, mediante el cultivo sumergido de hongos filamentosos. Desde el punto de vista hidrodinámico, estos procesos involucran fundamentalmente la dispersión de hasta cuatro fases diferentes: agua-aceite-aire-hongo. Aun cuando se sabe que la hidrodinámica del cultivo determina la eficiencia global del proceso de fermentación, poco se ha logrado en cuanto al entendimiento de las relaciones hidrodinámica-dispersión-fisiología-productividad. Esto se debe en parte a la falta de metodologías que permitan cuantificar y caracterizar con precisión tanto la viabilidad del hongo (ya que en buena medida determina el rendimiento y la productividad del metabolito de interés), como el tamaño de las burbujas de gas y de gotas de aceite (ya que de ello depende la eficiencia de transporte de nutrientes como el oxígeno y los ácidos grasos del aceite). En este trabajo se resume y revisa el desarrollo de las metodologías que nuestro grupo ha publicado, basadas en el procesamiento y el análisis digital de imágenes aplicadas al estudio del cultivo de Trichoderma harzianum, hongo que produce antimicóticos de alta potencia (como la 6-pentil-alfa-pirona). Con el uso de estas metodologías hemos generado conocimiento básico de los fenómenos que ocurren en el fermentador, lo que nos ha permitido establecer estrategias para incrementar la productividad de este tipo de procesos. Palabras clave: cultivos miceliares; hidrodinámica; dispersión multifásica; fisiología microbiana; productividad; análisis de imágene